4.2- Effect of BHRF1 on cell metabolism: Flux Balance Analysis
Metabolism of animal cells growing in high glucose concentration media
is characterized by their high rates of glycolysis fluxes. Glucose
metabolism of aerobic microorganisms yield pyruvate, which is partially
converted into acetyl-CoA and then can be completely oxidized to
CO2 and H2O in TCA cycle. However, in
mammalian cell lines, pyruvate is primarily converted into lactate
(Martínez-Monge et al., 2018a). Generation and accumulation of large
amounts of lactate, since glucose cannot be completely oxidized, is the
major consequence of such high glycolytic fluxes, leading to an
unbalanced metabolism. This metabolic behavior has been observed in many
hybridoma cell lines, regardless of the level of dissolved oxygen in
culture. Lactate generation from pyruvate seems to be necessary to
fulfill the NADH regeneration requirements in the cytoplasm (Mulukutla
et al., 2012), due to the limiting transport rates of NADH into
mitochondria, where can also be regenerated. Even though pyruvate
conversion to lactate is a much less energetically efficient process
than its oxidation in the Krebs cycle (Martínez et al. 2013).
Flux Balance Analysis (FBA) is a usefulness tool to obtain more
information about the redistribution of the internal metabolic fluxes
and could help to generate some hypothesis about the effects on
metabolism generated by BHRF1. Figures 4 and 5 show
the distribution of the metabolic fluxes using the genome-derived model
for Mus musculus detailed in the Calculations section. Metabolic
flux balances were conducted using the data obtained from the bioreactor
cultures.
FBA shows a deregulated metabolism in both cell lines, characterized by
high glycolytic fluxes and the consequent lactate generation and
secretion. However, glycolytic fluxes were reduced by 54% (bottom part
of glycolysis as a reference) due to the reduction in glucose
consumption (51%) in KB26.5-BHRF1. Lactate generation flux was dropped
to more than 60%. Despite the lower fluxes in glycolysis presented by
KB26.5-BHRF1 strain respect to KB26.5, the rate of carbon influx from
cytoplasm to mitochondria through pyruvate was 14% higher (320 versus
274 nmol/(mg·h)). In other words, when calculating the fluxes in mass
units (mg metabolite/(mg DCW ·h)) only 13% of the total glucose
consumed is entered to the mitochondria in KB26.5 cells, whereas it
increases up to 32% in the engineered KB26.5-BHRF1. When performing
similar calculations to estimate the lactate generation ratio regardless
the glucose consumption, lactate formation represents about 87% and
69% of the consumed glucose in KB26.5 and KB26.5-BHRF1, respectively.
Lactate generation in mammalian cell cultures is a well-known issue that
has been extensively studied (Hartley et al., 2018; Zagari et al.,
2013). At present, the most accepted hypothesis of why lactate is
generated argues in the regeneration of the reducing power (NADH) in the
cytoplasm, due to the high glycolytic fluxes (Hartley et al., 2018;
Zheng, 2012). To this end, there are two ways to regenerate NADH into
the cytoplasm: 1) pyruvate conversion to lactate and 2) Malate-Aspartate
Shuttle (Mulukutla et al., 2012). The interesting point is that
Malate-Aspartate Shuttle allows not only to regenerate NADH but also to
increase TCA cycle fluxes (importing malate), allowing then to generate
energy in form of ATP. On the other side, lactate generation provokes
the total loss of both carbon source and ATP generation. Then, the
reason for lactate generation could be found in a flux limit of
Malate-Aspartate Shuttle, leading the cells to generate lactate and
display this wasteful metabolism.
In the glucose breakdown to two pyruvate molecules, two molecules of ATP
and two of NADH are generated. Since the inner mitochondrial membrane is
impermeable to NADH, Malate-Aspartate Shuttle works as an indirect
transport system. It has been reported that the flux through this
transport occurs at lower rates than glycolysis (Schantz et al., 1986),
and the increased LDH activity is due to the inability of transporting
NADH through the shuttle at the same rates in which is generated. Under
conditions of increased cellular energy demand, higher glycolytic fluxes
are observed and, consequently, NADH production rates increases
proportionally. Such increase in NAD+ regeneration
needs is compensated by higher LDH activity in the cytosol, but not
differences in the Malate-Aspartate Shuttle fluxes are observed (Robergs
et al., 2004).
In this case, FBA shows that the rate of transport of reduction power
from cytoplasm to mitochondria through Malate-Aspartate Shuttle seems to
be a bit higher in KB26.5-BHRF1 than in KB26.5, showing a 19% of
increase, with a rate of 228 versus 192 nmol/(mg·h) in the
aspartate-glutamate mitochondrial symport transport. However, in both
cases the high glycolytic fluxes lead the cells to generate lactate, as
the Malate-Aspartate shuttle seems to not be enough powerful to couple
with the cytoplasmic NADH regeneration. In addition, a slight increase
in pathways related with the biomass formation is observed in
KB26.5-BHRF1, due to the higher growth rate; as Pentose Phosphate
Pathway to generate nucleotides or citrate export from the mitochondria
to lipids synthesis.
Due to the high growth rate in KB26.5-BHRF1, an increase in the TCA
fluxes could be expected, to generate more energy and therefore biomass.
However, comparing TCA cycles for both cell lines no significant
increase is observed, what is in accordance with the similar oxygen
consumption rate (Table 5 ). What it is clear from the FBA
performed is the evident collapse between glycolysis and TCA pathways in
both cases, as the high glucose uptake rate collapses the transport of
NADH to mitochondria, thus NADH should be regenerated into the cytoplasm
using pyruvate by means of lactate dehydrogenase.
The reduction of the glycolytic pathway in KB26.5-BHRF1 involves a
reduction of ATP production in the cytoplasm, as well as a reduction of
NADH formation. Consequently, the needs for oxidizing the NADH formed in
the cytoplasm also decreased and, together with the slightly higher
rates related to Malate-Aspartate Shuttle, yielded to a reduction of the
lactate formation. To further discuss the results, an analysis of the
synthesis and consumption of ATP was performed. Figure 6 shows
the distribution of the ATP formation (in regard to the reaction in
which ATP is generated) and its consumption.
The results show a significant reduction in the total ATP generation
and, as a consequence, ATP consumption in KB26.5-BHRF1 compared with the
parental cell line. As pointed out above, higher amounts of ATP were
synthetized along glycolysis in parental KB26.5 (proportionally to
glycolytic fluxes increase), but also an increase in the oxidative
phosphorylation pathway is observed, being very important for the
discussion for its high capacity of ATP generation. Regarding the ATP
consumption, KB26.5-BHRF1 needs more ATP to generate more biomass, which
also includes other pathways as lipids and nucleotide synthesis. The
most interesting part, and the key fact of this analysis, is the huge
difference in ATP requirements for cell maintenance. One possible
explanation of this maintenance reduction could be the higher efficient
substrate consumption and specially the lactate generation reduction of
KB26.5-BHRF1. As recently pointed out by Buchsteiner et al. (2018), the
energy requirements in lower lactate production cell lines may decrease
due to the lower energy requirements for maintaining ion gradients.
The results presented here suggest that the engineered KB26.5-BHRF1
hybridoma cell line somehow have altered such metabolism. BHRF1 is an
antiapoptotic protein located in the inner mitochondrial membrane
(Milian et al. 2015). Due to this fact, BHRF1 could be somehow affecting
carbon and NADH/NAD+ transport between cytoplasm and mitochondria. In
fact, most of the antiapoptotic genes reported in literature are known
to be bind at the mitochondrial membrane, regulating the apoptosis
through modulation of the mitochondrial permeability, but also playing
and important role in the metabolic processes of mitochondria (Majors et
al., 2007). Dorai et al. (2009) reported the effect of two antiapoptotic
genes in the metabolism of CHO, showing an important reduction in the
final lactate concentration due to the lactate consumption during the
culture. In addition, engineered cells showed a more efficient nutrient
consumption profile and less by-products generation, as ammonia or
alanine, as observed in this study.